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Immunofluorescence sample labeling detected by flow cytometry

2021-02-20
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Introduction:

Commonly used fluoresceins for flow cytometry detection include FITC, TRITC, Cy3, Cy5, PE and PI. Among the above various fluorescent dyes, PE has the strongest fluorescence and is suitable for weakly expressing antigens; FITC is the cheapest and is suitable for strong Express antigen, wide application range.

1

Flow cytometer (Flow cytometer) is a device for automatic analysis and sorting of cells. It can quickly measure, store, and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort out designated cell subgroups according to the preselected parameter range.

flow cytometry

Most flow cytometers are zero-resolution instruments. They can only measure indicators such as total nucleic acid amount and total protein amount of a cell, but cannot identify and measure the amount of nucleic acid or protein in a specific part. In other words, its detail resolution is zero.

Commonly used fluoresceins for flow cytometry detection include FITC, TRITC, Cy3, Cy5, PE and PI. Among the above various fluorescent dyes, PE has the strongest fluorescence and is suitable for weakly expressing antigens; FITC is the cheapest and is suitable for strong Express antigen, wide application range.

  1. Direct immunofluorescence labeling
    Take a certain amount of cell suspension (concentration about l×106 cells/m1), and directly add fluorescein-labeled antibody for immunoreaction (such as double labeling or multi-labeling staining, several antibodies labeled with different fluorescein can be added at the same time ). After incubating at 4°C for 15-30 minutes, wash with l/15mol/L PBS (pH 7.2-7.4) for 1-2 times, add buffer to resuspend, and test on the machine.
    This method is simple to operate, accurate results, easy to analyze, and is suitable for simultaneous determination of multiple parameters in the same cell population. Although direct fluorescent antibody labeling reagents cost more. However, the strong non-specific fluorescence interference in the indirect labeling method is reduced, so it is more suitable for the detection of clinical specimens.

  2. Indirect immunofluorescence labeling
    Take a certain amount of cell suspension (concentration about 5×106 cells/m1), add specific primary antibody, and incubate at 4°C for 15-30 minutes. After the reaction is complete, wash out unbound antibody with phosphate buffer and absorb the remaining Add the fluorescently labeled secondary antibody to the liquid and incubate at 4~C for 30 minutes to generate an antigen-antibody-anti-antibody complex, which can be tested on the machine after washing. Note: In the entire fluorescent labeling process, you need to refer to the immunofluorescence cytochemical indirect staining procedure, especially the normal non-immune serum should be used to block before adding the primary antibody to reduce non-specific reactions. If you need to use 0.3% TritonX-100 to perforate the cell membrane for unfixed cells to ensure that the antibody can enter the cell. The cost of this method is low, the secondary antibody is widely used, and it is mostly used for scientific research sample detection. However, due to the strong non-specific fluorescence background, which affects the experimental results, negative or positive controls should be added during sample preparation. In addition, because the indirect method has many steps, especially after multiple washings, the number of cells can be drastically reduced. Therefore, the cell concentration of the cell suspension is about 5×10′ cells/ml before the first antibody is added. No less than 1×106 cells/ml is sufficient. This method is not suitable for measuring samples with a small number of cells.

  3. Precautions in fluorescent labeling
    (1) In order to reduce non-specific interference, the fluorescent label should be filtered with 0.22um filter membrane or centrifuged at high speed for 5 minutes before use to remove particles or sediment.
    (2) The preparation, staining and storage of cell samples should be kept as fresh as possible to prevent the disappearance of surface antigens and cell death caused by catabolism. It can be done by adding nutrient media or low-concentration serum and 4~0 or ice bath operation. solve.
    (3) In order to ensure accuracy, dead cells should be excluded in the analysis of sparse cells or low proportion of cell subpopulations.
    (4) The concentration of fluorescently labeled antibody should be appropriate. If the concentration is too high, non-specific binding will increase. Before using the primary antibody, incubate the sample with excess bovine serum albumin (BSA) or normal serum derived from the same species to block the non-specific effects of the primary antibody on the cell surface or intracellular structure. After using the primary antibody, incubate the sample, 5%-10% normal serum of the same species as the secondary antibody, and the secondary antibody to reduce non-specific interactions between the secondary antibody and the primary antibody, cell surface or intracellular structure . If the antibody is diluted with a serum liquid containing the same species as the primary or secondary antibody, this step can be omitted.

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